Genetic indicators for imaging of cell signaling in plasticity and development

描述（由申请人提供）：在过去的十年中，在成像、分子生物学和生物化学方面取得了惊人的进展，使单个蛋白质在体内的行为可视化。在这里，我建议发展和可视化的时间和空间动态的细胞内信号在活的神经元。正如钙成像的早期工作通过定义其空间和时间特征重新定义了我们对钙流入重要性的理解，我相信可视化细胞内信号的空间和时间动态将对我们对神经系统的理解有类似的好处。最初，我们已经开发了能够可视化许多细胞内信号级联的关键第一步之一的指标：酪氨酸磷酸化。在过去的几年里，我们已经开发了一个系统，依赖于比例成像的变化，在磷酸化的遗传编码荧光指标。我们现在提出三个具体目标，将这些工具开发成一个监测神经元可塑性和发育过程中的信号的系统。我们建议：1)建立一个有针对性的指标库来报告特定激酶的活性；2)开发定位于特定细胞区室的指标；3)制定指标，同时报告多个信号分子的活性。使用我们的指示器，工作人员将能够阐明突触可塑性背后的信号动力学。因此，我们的工具将使人们对神经元可塑性的基本机制有新的认识。神经可塑性是大脑许多基本功能的基础，而异常的神经可塑性与疾病有关。过度的可塑性可能是癫痫和成瘾等疾病的基础，而可塑性缺陷可能在癫痫、神经退行性疾病和自闭症谱系障碍中发挥重要作用。我们的研究将通过开发新的工具，以亚细胞分辨率可视化动态神经元信号，在所有这些水平上产生广泛的影响。

DESCRIPTION (provided by applicant): During the past decade stunning advances have been made in imaging, molecular biology and biochemistry that enable the visualization of the behavior of single proteins in vivo. Here, I propose to develop and visualize the temporal and spatial dynamics of intracellular signaling within living neurons. Much as early work in calcium imaging redefined our understanding of the importance of calcium influx by defining its spatial and temporal characteristics, I believe visualizing the spatial and temporal dynamics of intracellular signaling will have similar benefits to our understanding of the nervous system. Initially we have developed indicators that enable visualization of one of the key first steps in many intracellular signaling cascades: tyrosine phosphorylation. During the past several years, we have developed a system that relies on ratiometric imaging of changes in a genetically encoded fluorescent indicator of phosphorylation. We now propose three specific aims to develop these tools into a system for monitoring signaling during neuronal plasticity and development. We propose to: 1) Develop a library of indicators targeted to report activity of specific kinases; 2) Develop indicators that localize to specific cellular compartments; 3) Develop indicators to report activity of multiple signaling molecules simultaneously. Using our indicators, workers will be able to elucidate the dynamics of signals that underlie synaptic plasticity. Thus, our tools will enable novel insights into essential mechanisms that underlie neuronal plasticity. PUBLIC HEALTH RELEVANCE: Project Narrative Neuronal plasticity underlies many fundamental functions within the brain, while abnormal neuronal plasticity is associated with disease. Excessive plasticity may underlie diseases like epilepsy and addiction, while defects in plasticity could play important roles in epilepsy, neurodegenerative, and autism spectrum disorders. Our research will have broad impacts across all these levels by developing new tools to visualize dynamic neuronal signaling with subcellular resolution.